Rhabdomyosarcoma is the most common soft-tissue sarcoma occurring in children and young adults. These tumors are unique among sarcomas in that they are thought to derive from skeletal muscle tissue. Tumors express myogenic markers, but fail to undergo terminal differentiation. Two subclasses of rhabdomyosarcomas exist, the embryonal and alveolar. Whereas the embryonal form compromises the majority of cases and is more responsive to multi-modal therapy, alveolar rhabdomyosarcoma is a more primitive malignant neoplasm where prognosis for children with advanced disease remains dismal. Therefore, effective therapies will likely hinge on our abilities to better resolve the underlying mechanisms of alveolar rhabdomyosarcoma. NF-kappaB is a transcription factor whose signaling pathway has been implicated in a variety of cancers. In skeletal muscle, NF-kappaB functions in immature cells to negatively regulate terminal differentiation. Recently we discovered that NF-kappaB functions in this capacity by participating in a regulatory circuit with the Polycomb group protein, YY1, and the microRNA, miR-29. In undifferentiated myoblasts, NF-kappaB stimulates YY1 production, which epigenetically silences miR-29. However, as differentiation ensues, NF-kappaB activity decreases causing a concomitant reduction of YY1 and a derepression of miR-29. This microRNA in turn feeds back on YY1 to further reduce its expression and favor a differentiation state. Significantly, in rhabdomyosarcoma cell lines and primary tumors NF-kappaB - YY1 - miR-29 circuitry becomes deregulated as a result of elevated levels of NF-kappaB that cause silencing of miR-29. Further results indicate that NF-kappaB activation in rhabdomyosarcoma is mediated through the NF-kappaB classical pathway that switches from the alternative pathway found in normal differentiated muscle. Inhibition of classical NF-kappaB signaling in rhabdomyosarcoma cells restores the regulatory circuit and promotes a muscle differentiation phenotype. Based on these results we hypothesize that NF-kappaB contributes to rhabdomyosarcoma by blocking terminal differentiation through deregulation of the NF-kappaB - YY1 - miR-29 circuit. To test this hypothesis, specific aims will 1) determine the relevance of the NF-kappaB classical versus alternative signaling pathway in rhabdomyosarcomagenesis, and 2) determine the requirement of NF-kappaB in the initiation and progression of alveolar rhabdomyosarcoma. Results from these studies are likely to yield significant mechanistic insight into the role of NF-kappaB signaling in alveolar rhabdomyosarcoma, which may prove useful for treatment of this lethal childhood cancer.